Technical Field
The present disclosure relates, generally, to synthetic antisense oligonucleotides (AON) and methods employing antisense oligonucleotides for modifying splicing events that occur during pre-mRNA processing or for down-regulating the expression of mutated mRNA that contain repeated sequences such as, for example, 3′ or 5′ CUG, CAG, and/or CCUG. More specifically, disclosed herein are tricyclo-DNA (tc-DNA) AON that are effective in facilitating exon skipping during pre-mRNA processing, in masking intronic silencer sequences and/or stem-loop sequences in pre-mRNA, and in targeting the RNase-mediated destruction of mRNA. Described herein are tc-DNA AON that may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping mutated exons, such as a mutated exon 23 or exon 51, within a dystrophin gene to restore functionality of a dystrophin protein. Also described are tc-DNA AON that may be used in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein. Still further tc-DNA AON described herein may be used in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats. Thus, tc-DNA AON and one or more of the foregoing approaches can be used to restore functionality in a protein involved in a myopathy.
Description of the Related Art
Duchenne Muscular Dystrophy (DMD) is the most common hereditary myopathy, afflicting about one in 3,500 males regardless of ethnicity. Although infrequent, girls and women may present Duchenne-like symptoms in manifesting carriers. The foremost consequence of DMD is that muscle fibers become particularly fragile and natural muscle activity provokes general damage in muscle tissue. The end-point observed in DMD, as well as in many muscle dystrophies, is that slow degeneration leads to almost complete fibrosis with fatty infiltration. Because of spine deformation and breathing difficulties, life expectancy in the 1960s was about 15 years. In the absence of cardiac complications, modern improvements in management methods (i.e. arthrodesis and tracheotomy ventilation) have increased life expectancy to 30 years.
Clinical symptoms of DMD are evident at the age of 18 months to three years and include a delayed ability to walk and climb, difficulty getting up from the floor, and abnormally enlarged calves. At about 5 to 6 years, muscle contractions develop in the foot, knee, and hip joints. Progression of the disease is characterized by a continual muscle wasting, leading at about 9 to 12 years to the loss of walking ability. In addition, some Duchenne boys present mental retardation suggesting that the missing protein is also involved in the central nervous system.
Duchenne Muscular Dystrophy is an X-linked recessive disorder. The DMD locus was identified on the X-chromosome (Xp21.2-OMIMid: 310200) in 1986, through a positional cloning approach, in a gene that encodes a protein called dystrophin. Mutations in the dystrophin gene result in a failure to produce dystrophin in striated muscles. Mothers of affected boys have a two-thirds chance of carrying a dystrophin mutation, while approximately one-third of patients have de novo mutations. More than half of DMD boys exhibit large genomic deletions encompassing one to several exons; few of them have large sequence duplications. Others have point mutations or very small deletions or duplications that are difficult to identify.
The extent of the mutations does not, however, directly correlate with the severity of the phenotype. Out-of-frame deletions or non-sense mutations that yield premature stop codons and subsequent abortion of translation result in dystrophin deficiencies characterized by severe phenotypes. In-frame deletions are responsible for a milder myopathy known as Becker muscular dystrophy (BMD).
With nearly 2.5 million base pairs, the DMD locus is the longest gene ever detected, but only about 14,000 base pairs contain coding sequences, which arc spread over 79 exons. Full length dystrophin (DP 427) is a 427 kDa cytoskeletal protein expressed in all muscles, but a variety of protein isoforms (DP 260, DP 140, DP 116, DP 71) are generated by the tissue-specific, differential usage (in the retina, central nervous system, peripheral nervous system, and non-muscle tissues) of four internal promoters located in introns 29, 43, 55, and 62, respectively.
Full-length dystrophin is an essential component of a sarcolemmal glycoprotein complex (SGC) involved in sustaining the membrane integrity of muscle fibers by linking myofiber cytoskeleton to the extracellular matrix. Sequence analysis has predicted that the dystrophin protein entails several domains and repeats. Schematically, there is an actin-hybridizing site at the N-terminus (N-ABD); a central rod domain (RD; having 24 spectrin-like repeats) containing four hinge segments (H) that may confer flexibility; and a cystein-rich domain (CRD), which binds other members of the DPC, near the C-terminus (CT).
Structure/function analysis has identified domains which are crucial for protein function. This was exemplified by internal deletions occurring in some patients with a mild disease in whom the deletion encompassed exons 17 to 48 (46% of the coding sequence). England et al., Nature 343(6254):180-2 (1990). This led to the concept of functional “minidystrophin” extensively used in the past 10 years in gene transfer experiments. It is now established that removal of the N-ABD and CT domains cause moderate loss of function, while the CRD is essential. Alterations of the RD result in diverse phenotypes depending on the extent and nature of the truncation. As an example, an RD deleted dystrophin (ΔR1-R24) is not functional, whereas a (ΔH2-R19) truncated dystrophin, which retains eight complete spectrin-like repeats out of 24, results in a protein with full activity.
There are two well-characterized genetic animal models for Duchenne Muscular Dystrophy. The mdx mouse harbors a non-sense mutation in exon 23 of the dystrophin gene, which precludes the synthesis of full-length, wild-type dystrophin protein. The mdx mouse displays a compensatory mechanism counteracting the degeneration, which could maintain the regeneration process to restore the mechanical damage. The mdx mouse does not exhibit symptoms of DMD and its life span is almost normal.
The GRMD (Golden Retriever Muscle Dystrophy) dog lacks functional dystrophin because of a splice site mutation in intron 6, which disrupts the reading frame. In GRMD, as with human DMD, the progressive degradation of fibers leads inexorably to skeletal musculature wasting with marked endomysial and perimysial fibrosis. Because of its DMD-like phenotype, GRMD remains the best available model for the evaluation of potential therapies for DMD.
Despite the identification and characterization of mutations in the dystrophin gene that are associated with an onset of DMD and the availability of suitable animal model systems for testing prospective therapeutic agents, there remains a need in the art for compositions and methods for the treatment of this disease. Several studies over the past 10 years support the benefit of steroid treatment (prednisone and deflazacort) in Duchenne boys, although a broad statistical evaluation has not yet been fully completed. Pharmacologic-induced read-through of premature stop-codon mutations by means of gentamicin medication could also potentially be effective in up to 5% of patients with DMD. Clinical trials are being carried out in the United States and Italy, even though the results of preclinical studies in the mdx mouse model were controversial. A new drug (PTC124) developed by PTC Therapeutics seems more promising. Studies arc also underway to upregulate the utrophin gene using drugs whose product, the dystrophin-like protein utrophin, can compensate for the function of the missing dystrophin.
There are many other avenues of research; as an example, it has been recently shown that antagonizing myostatin by using blocking antibodies could improve muscle strength in mdx mice. This approach was initially based on multiple injections of normal myoblasts into the diseased muscles. Partridge et al., Nature 337(6203):176-9 (1989). Subsequent clinical trials (1991-98) have failed, although improving cell manufacturing and delivery procedures have made possible a new phase I trial in Canada (2002). Recent developments have also provided evidence that stem cells from either bone marrow or vascular origins can target skeletal muscle through the systemic pathway, even though the extent of the genetic correction is still insufficient.
Gene therapy for DMD lies on in situ delivery of dystrophin mini-genes into skeletal fibers by using gene vectors as vehicles. A first exploratory study using naked full length cDNA in a plasmid vector was carried out in France (2000-03). Among the different types of vectors that have been tested for muscle gene therapy, adenovirus associated virus (AAV)-derived vectors seem to be the most promising. AAV vectors have a number of advantages: (i) they arc able to infect a wide variety of cell types including muscle fibers; (ii) they appear safe because they lack all viral genes and that wild type viruses have not yet been associated with any pathology in human; (iii) conversely to wild type AAVs, which integrate into the genome of the host cells, replication deficient AAV vectors generally persist as episomes thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (iv) in contrast to other vector systems, AAV vectors do not trigger a significant immune response thus granting long term expression of the therapeutic transgenes (provided their gene products were not rejected). AAV vectors can also be produced at high titer and forced intra-arterial injections make them able to achieve gene transfer to significant muscle territories through a single injection, at least in rodents. Although AAV vectors lack all viral genes, their cargo shipment is limited to 4.5 kb. For that reason, the choice of AAV led to the development of μ-dystrophin variants of about 4 kb instead of the full-length dystrophin (14 kb). Several of these variants have been beneficially tested in the mdx model by either transgenesis or gene transfer.
In many DMD patients as well as in the mdx mouse and the GRMD dog, rare dystrophin-positive fibers have been reported. Although the proportion of revertant fibers increases with time, their number is unfortunately too low to confer a significant clinical benefit. The mechanism initiating these revertant fibers remains unknown although studies suggest that the reading-frame may be restored by exon-skipping. Such a natural phenomenon has prompted investigation into the design of strategies for gene repair/modulation based on the use of 2′-O-methyl antisense oligoribonucleotides as well as Morpholinos to interfere with splicing, thus inducing exon skipping. Indeed, this approach has been successfully used in vitro in mdx, GRMD and DMD muscle cells as well as in vivo (successful phase 1 clinical trial for 2′-O-methyl in Netherlands; a phase 1 with Morpholinos is ongoing in UK). Nevertheless, the weakness of this approach is that it requires regular administration of the synthetic AOs, and systemic delivery has not been fully achieved.
An alternative approach is to synthesize the sequences of interest in situ from vectors as antisense RNA molecules. Even so, producing “therapeutic” antisense RNA molecules in vivo poses many problems such as stability and subcellular localization. Small nuclear RNAs (snRNAs), which are known to participate in the splicing reaction, may be used as carriers to overcome these limitations. Recent reports have shown that U7 snRNA carrying antisense sequences against the splice junctions of either exon 23 or exon 51 of the dystrophin gene induce dystrophin synthesis in vitro as well as in vivo in mdx and Δ48-50 DMD cells, respectively.
An in silico search of all DMD patients with an out-of-frame deletion who would theoretically benefit from the skipping of a single exon adjacent to the deletion (on either side) has been performed. Interestingly, it is predicted that skipping exon 51 should restore a mini-dystrophin in 22% of the cases (i.e. Δ45-50, Δ47-50, Δ48-50, Δ49-50, Δ50 and Δ52). The resulting truncated proteins are expected to be at least partially functional since they correspond to deletions that have been found in some BMD patients. Additionally, a few healthy males carrying Δ51-52 and Δ48-51 in-frame deletions have been identified. Skipping of exon 51, in select patients, should bring about the production of a functional shorter dystrophin thus improving the phenotype.
Mental retardation is a symptom frequently associated with DMD and can result from the lack of dystrophin in neuronal cells. Rescuing a semi functional dystrophin in the brain could therefore correct or improve the cognitive impairment.
Spinal Muscular Atrophy (SMA) refers, generally, to a variety of disorders deriving from a common genetic defect in a survival motor neuron (SMN) gene, which, in 1990, was mapped to chromosome 5q11.2-13.3. Human chromosome 5 contains a large duplication such that there arc two copies of the SMN gene, SMN1 and SMN2.
SMA is the most common cause of genetically determined neonatal death. All forms of SMN-associated SMA have a combined incidence of about 1 in 6,000. The gene frequency is around 1:80 and approximately one in 40 persons is a carrier. There are no known health consequences of being a carrier and the only way one may know to consider the possibility is if a relative is affected.
SMA is characterized by the loss of the motor neurons of the spinal cord and brainstem. In general, the earlier the symptoms appear, the shorter the expected life-span. Once symptoms appear, the motor neuron cells quickly deteriorate. All forms of SMA have in common weakness caused by denervation, that is, the muscle atrophies because it has lost the signal to contract due to loss of the innervating nerve. Spinal muscular atrophy only affects motor nerves. Heritable disorders that cause both weakness due to motor denervation along with sensory impairment due to sensory denervation are known by the inclusive label Charcot-Marie-Tooth or Hereditary Motor Sensory Neuropathy.
The course of SMA is directly related to the severity of weakness. Infants with the severe form of SMA frequently succumb to respiratory disease due to weakness of the muscles that support breathing. Children with milder forms of SMA naturally live much longer although they may need extensive medical support, especially those at the more severe end of the spectrum.
Type I SMA, also known as severe infantile SMA or Werdnig Hoffmann disease, is the most severe, and manifests in the first year of life. This type generally onsets quickly and unexpectedly after birth; babies diagnosed with Type I SMA do not generally live past one year of age. Pneumonia is considered the ultimate cause of death due to deterioration of survival motor neurons; motor neuron death causes insufficient functioning of the major bodily organ systems, particularly respiratory (e.g., breathing and ridding of pooled secretions inside lungs). Type II SMA, or intermediate SMA, describes those children who are never able to stand and walk, but who are able to maintain a sitting position at least some time in their life. The onset of weakness is usually recognized some time between 6 and 18 months. Weakness slowly and gradually increases over the life of the individual. Type III SMA patients are able to walk at some time.
SMA is typically diagnosed with a survival motor neuron (SMN) gene test, which determines whether there is at least one copy of a functional SMN1 gene, which is distinguished from the highly similar SMN2 gene, by the presence of exons 7 and 8 in fully-processed mRNA. The SMN2 gene also contains a mutation that makes it less efficient at making protein, though it does so in a low level. SMA is caused by loss of the SMN1 gene from both chromosomes and the inability of SMN2 protein to compensate for the loss in functional SMN1 protein.
Current strategies for developing SMA therapeutics include identifying drugs that increase SMN2 levels, enhance residual SMN2 function, or otherwise compensate for the loss of SMN1 activity. Drugs such as butyrates, valproic acid, hydroxyurea, and riluzole (Rilutek®, Sanofi Aventis) are or have been under clinical investigation for the treatment of SMA. Although gene replacement strategies are being tested in animals, current treatment for SMA consists of prevention and management of the secondary effect of chronic motor unit loss. There is currently no drug known to alter the course of SMA and it is likely that gene replacement for SMA will require many more years of investigation before it can be applied to humans.
Myotonic Dystrophy (DM) is a chronic, slowly progressing, highly variable inherited multisystemic disease that can manifest at any age from birth to old age. Myotonic dystrophy is the most common form of adult onset muscular dystrophy and the second most common form of any skeletal muscle disease after Duchenne muscular dystrophy. DM is characterized by wasting of the muscles (muscular dystrophy), posterior subcapsular iridescent cataracts (opacity of the lens of the eyes), heart conduction defects, endocrine changes and myotonia (difficulty relaxing a muscle).
There are currently two known types of adult onset DM, both identifiable by DNA analysis: Myotonic dystrophy type 1 (DM1) is commonly referred to as Steinert's disease, which has a congenital form that can severely affect babies and a childhood onset form. Myotonic dystrophy type 2 (DM2) is known as PROMM or proximal myotonic myopathy. Additional forms of myotonic dystrophy (e.g., DM3, DM4, DMX) are suspected, but their existence remains unproven. While both DM1 and DM2 are considered to be slowly degenerative conditions, DM2 is considered to be generally milder than DM1.
Presentation of symptoms varies considerably by form (DM1/DM2), severity and even unusual DM2 phenotypes. DM1 patients often present with myotonia, disabling distal weakness and severe cognitive problems. DM2 patients commonly present with muscle pain, stiffness, fatigue, or the development of proximal lower extremity weakness. Day et al. Neurology 60(4): 657-64 (2003). The characteristic pattern of weakness is different for DM1 and DM2. In DM1, it is noted in face and jaw muscles, the drooping of the eyelids (ptosis), weakness of the neck muscles, hands and lower legs. In DM2, the weakness is more evident in proximal muscles, those closer to the trunk of the body, neck, shoulders, hip flexors and upper legs.
DM1 symptoms include hypersomnia (daytime sleepiness), muscle wasting, dysphagia, and respiratory insufficiency. DM1 patients may experience a more diverse range of cognitive problems than DM2 patients. Depending on what form they have and the degree of severity, DM1 cognitive problems may range from developmental delays, learning problems, language, speech, behavior, apathy, or hypersomnia. Cognitive manifestations for DM2 include problems with executive function (i.e. organization, concentration, word-finding etc.) and hypersomnia.
In DM1, the affected gene is called DMPK (myotonic dystrophy protein kinase) and codes for a serine/threonine protein kinase expressed in skeletal muscle. The gene is located on the long arm of chromosome 19. In DM1, the DMPK gene is characterized by a triplet repeat of Cytosine-Thymine-Guanine (CTG). The number of repeats varies greatly from person to person, but the average number in a healthy person is between 5 and 37. Sometimes when repetitive sequences of DNA are repaired or replicated during cell division, the cellular machinery slips and an extra copy of the triplet repeat is added to the sequence. Once there are more than 37 triplet repeats in the DMPK gene the sequence becomes unstable and slippage becomes more common.
People affected with DM1 have over 50 and can have as many as 2000 CTG repeats. The result being that the repeat size of an individual with DM1 will become larger usually during gametogenesis or early embryonic development, such that children of an affected adult typically exhibit larger expansions than their parent due to slippage during gametogenesis (this phenomenon is referred to as anticipation). Individuals with larger expansions have an earlier onset of the disorder and a more severe phenotype.
DM2 is similarly caused by a defect of the ZNF9 gene on chromosome 3q21. The repeat expansion for DM2 is much larger than for DM1, ranging from 75 to over 11,000 repeats and involves a repeat of four nucleotides. Unlike DM1, however, the size of the repeated DNA expansion does not appear to make a difference in the age of onset or disease severity in DM2. Anticipation appears to be less significant in DM2.
There is currently no cure for or treatment specific to myotonic dystrophy. Heart problems, cataracts, and other abnormalities associated with the condition can be treated but not cured. There are, however, medical interventions and medications that may relieve some of the symptoms such as myotonia, pain, and excessive sleepiness. Research in areas such as high throughput screening and antisense therapy hold hope for more effective targeted treatments for the future. Altered splicing of the muscle-specific chloride channel 1 (C1C-1) causes the myotonic phenotype of DM1 and is reversible in mouse models using Morpholino antisense oligonucleotides that modify the splicing of C1C-1 mRNA. Wheeler et al., J. Clin. Invest. 117(12):3952-7 (2007).
Despite the ongoing search for therapeutic modalities for Duchenne Muscular Dystrophy, Spinal Muscular Atrophy, and Steinert's Myotonic Dystrophy, there remains an urgent need for efficacious compounds and therapeutic methods for the treatment of these diseases.